Neuropeptides are packaged in dense-core vesicles (DCVs) and released to control development, mood and behavior. Despite the importance of neuropeptides, there is only rudimentary knowledge of how DCVs are delivered to nerve terminals and undergo release in response to bursts of activity. Furthermore, it remains unclear how presynaptic signaling controls exocytosis of DCVs and small synaptic vesicles (SSVs), which contain classical transmitters. This proposal will continue to use GFP-based imaging of DCVs, Ca2+ and presynaptic regulatory and scaffold proteins to study maintenance and modulation of native nerve terminals in Drosophila, an experimental system that is well suited for genetics, electrophysiology and imaging in native intact synapses. Proposed experiments are based on: (a) our development of a new method for tracking single DCVs in native neurons for minutes (SPAIM), (b) the discovery of a new mechanism for delivering DCVs to sequential synaptic release sites called vesicle circulation, (c) the demonstration that synaptic neuropeptide release can be evoked by cAMP without Ca2+ influx, (d) findings suggesting that inositol trisphosphate receptors (IP3Rs) control translocation of regulatory and scaffold proteins in the nerve terminal, and (e) the ability to directly monitor activity-dependent release from a single vesicle in a native synapse for the first time. Here three main questions will be addressed with GFP-based imaging in Drosophila neurons: 1. How does vesicle circulation vary with nerve terminal shape, disease and acute injury? 2. How do single DCVs release their contents in response to different stimuli and why is release reduced with inhibition of retrograde transport, a condition associated with neurodegenerative diseases? 3. How does presynaptic signaling induce translocation of regulatory proteins within the nerve terminal? These studies will yield fundamental insights into the maintenance of terminals and the regulation of release of neuropeptides and their cotransmitters. Furthermore, the proposal will reveal how these processes are affected by acute nerve injury and diseases such as Fragile X syndrome and Lou Gehrig's disease (ALS).